Thermostable xylanase from a strain of Rhodothermus marinus

ABSTRACT

A xylanase obtained from Rhodothermus marinus, the method of obtaining it and a process for its use are disclosed. The xylanase has an optimum temperature of from 85° to 100° C., a relative activity of more than 50% in the interval of from pH 5 tp pH 8 after incubation for 5 minutes at 65° C., a relative temperature stability at 80° C. of more than 80% after incubation at pH 7 for 3 hours, a pH optimum of about 6 and an isoelectric point in the range of from 3 to 7. The xylanase can be obtained from the strains ATCC 43812, ATCC 43813 or a mutant thereof. The process for obtaining the xylanase involves cultivation of a xylanase-producing strain of Rhodothermus marinus in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts followed by recovery of the xylanase. Also disclosed is a process for the treatment of lignocellulosic pulp with the xylanase.

This application is a continuation of PCT/DK92/00300 filed Oct. 14,1992, the contents of which are incorporated herein by reference.

TECHNICAL FIELD

This invention relates to novel enzymes. More specifically, theinvention provides novel xylanases obtainable from strains belonging tothe genus Rhodothermus. The invention also relates to the use of thexylanases in the treatment of lignocellulosic pulp.

BACKGROUND ART

Xylan, a major component of plant hemicellulose, is a polymer ofD-xylose linked by beta-1,4-xyiosidic bonds. Xylan can be degraded toxylose and xylo-oligomers by acid or enzymatic hydrolysis. Enzymatichydrolysis of xylan produces free sugars without the by-products formedwith acid (e.g. furans).

Major applications for xylanases are enzymatic breakdown of agriculturalwastes for production of alcohol fuels, enzymatic treatment of animalfeeds to release free pentose sugars, manufacturing of dissolving pulpsyielding cellulose, and bio-bleaching of wood pulp [Detroyrn R. W. In:Organic Chemicals from Biomass, (CRC Press, Boca Raton, Fla., 1981)19-41.; Paice, M. G., and L. Jurasek., J. Wood Chem. Technol.4:187-198.; Pommier, J. C., J. L. Fuentes, G. Gorna., Tappi Journal(1989):187-191.; Senior, D. J., et al., Biotechnol. Letters 10(1988):907-912].

The pulp and paper industry is using xylanase compositions in thebleaching process to enhance the brightness of bleached pulps, todecrease the amount of chlorine used in the bleaching stages, and toincrease the freeness of pulps in the recycled paper process [Eriksson,K. E. L., Wood Science and Technology 24 (1990);79-101.; Paice, M. G.,R. Bemier, and L. Jurasek, Biotechnol. and Bioeng. 32 (1988):235-239.;Pommier, J. C., J. L. Fuentes, and G. Gorna, Tappi Journal(1989):187-191].

Kraft pulping, a process widely used in the pulp and paper industry,involves the alkaline sulfate cooking of pulp to remove 95% of thelignin. The remaining 5% of lignin gives the pulp a dark brown colourwhich has the tendency to darken in UV light or by oxidation. In orderto obtain a white pulp for high quality paper, the brown colour isremoved by a multi-stage bleaching process using chlorine and/orchlorine dioxide.

Presently, there is much concern about the environmental impact of thechemicals generated from the bleaching process. Enzymes can aid in theremoval of lignin from the pulp without any harmful side products.Reports show that lignin in wood is linked to xylan [Eriksson, O., etal., Wood Sci.Technol. 14 (1980);267.; Takashi, N., and T. Koshijiima,Wood Sci.Technol. 22 (1988);177-189]. By a limited hydrolysis of thexylan a greater release of lignin occurs during bleaching. Thus, byenzymatically treating the pulp prior to bleaching the amount of activechlorine needed would in turn decrease [Viikari, L., et al., Proceedingsof the 3rd International Symposium on Biotechnology in the Pulp andPaper Industry (1986);67].

SUMMARY OF THE INVENTION

Recently novel thermophilic bacteria named Rhodothermus have beenisolated from an alkaline hot spring in Iceland [Alfredsson, G. A.;Kristjansson, J. K.; Hjorleifsdottir, S.; Stetter, K. O. (1988):Rhodothermus marinus, gen.nov., sp.nov., a thermophilic, halophilicbacterium from submarine hot springs in Iceland; J. Gen. Microbiol. 134;299-306]. We have now found that these bacteria produce highlythermostable xylanolytic enzymes with good stability in a broad pHrange.

Accordingly, in its first aspect, the invention provides xylanaseshaving activity at temperatures of from below 60° to above 100° C., arelative activity of more than 50% in the interval of from pH 5 to pH 8after incubation for 5 minutes at 65° C., a relative temperaturestability at 80° C. of more than 80% after incubation at pH 7 for 3hours, being capable of hydrolysing birchwood xylans, and havingimmunochemical properties identical or partially identical to those of axylanase derived from the strain ATCC 43812, or the strain ATCC 43813,or the strain DSM 4252.

In another aspect, the invention provides a process for the preparationof a xylanase of the invention, which process comprises cultivation of axylanase producing strain of the genus Rhodothermus in a suitablenutrient medium, containing carbon and nitrogen sources and inorganicsalts, followed by recovery of the desired enzyme.

in a further aspect, the invention provides a process for treatment oflignocellulosic pulp, in which the lignocellulosic pulp is treated withan enzyme of the invention.

BRIEF DESCRIPTION OF DRAWINGS

The present invention is further illustrated by reference to theaccompanying drawings, in which:

FIG. 1 illustrates the temperature stability of a xylanase of theinvention, presented as % relative residual activity vs. time, atdifferent temperatures (□80° C.; 90° C.; 100° C.) and pH 7;

FIG. 2 illustrates the temperature optimum of a xylanase of theinvention, presented as specific xylanase activity vs. different 5 min.incubation temperatures, and pH 7;

FIG. 3 illustrates the pH stability of a xylanase of the invention,presented as absorbance at 540 nm (i.e. xylanase activity) vs. differentpH values, and at different incubation times (□0 h inc.; 4 h inc.; 8 hinc.), and 65° C.; and

FIG. 4 illustrates the pH activity of a xylanase of the invention, afterincubation for 5 min. at 65° C., presented as the absorbance at 540 nMmeasured at pH values 4-11.

DETAILED DISCLOSURE OF THE INVENTION

The Organism

Rhodothermus marinus, representative of the genus Rhodothermus, is athermophilic bacterial strain that can be isolated in submarine alkalinehot springs in Iceland. This organism is aerobic, heterotroph,gram-negative, rod-shaped and non-motile. The strain is red-pigmented,salt dependent for growth and exhibits extracellular xylanase activity.

Two strains representative for Rhodothermus marinus have been depositedas type cultures and hence are publicly available from Deutche Sammlungvon Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-3300Braunschweig, Germany, under the accession number DSM 4252, and/or fromAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA, under the accession Nos. ATCC 43812 and ATCC 43813. Thedeposit ATCC 43812 is stated as being identical to the deposit DSM 4252.

The organisms are able to grow on agar plates and in liquid medium withthe appropriate supplementation, e.g. a medium as described inExample 1. Extracellular xylanase activity can be induced by xylan. Thechoice of culture system greatly affects the specific growth rate, andgrowth in complex or synthetic media also affects the productivity.

The Enzymes

The xylanases of this invention are obtainable from members of the genusRhodothermus, and they may be produced by cultivation of a strainbelonging to the genus Rhodothermus, preferably a strain of Rhodothermusmarinus, most preferred the strains ATCC 43813 and ATCC 43812, thelatter being identical to the strain DSM 4252, or mutants or variantsthereof, in a suitable nutrient medium, containing carbon and nitrogensources and inorganic salts, followed by recovery of the desired enzyme.The enzyme can also be obtained by recombinant DNA-technology.

The xylanase of the invention can be described by the followingcharacteristics.

Physical-Chemical Properties

The enzyme of the invention possesses xylanolytic activity attemperatures of from below 60° to above 100° C. No pronouncedtemperature optimum has been detected, but apparently (cf. FIG. 2) theenzyme possesses optimum activity within a broad temperature range offrom 80° C. to above 100° C., more specifically the range of from 85° C.to 100° C.

The enzyme of the invention has a relative activity of more than 50% inthe interval of from pH 5 to pH 8 after incubation for 15 minutes at 65°C. Moreover, it appears (cf. FIG. 4) that the pH optimum of the enzymeis in the range of from pH 5 to 7, more specifically around pH 6, asmeasured after incubation for 5 minutes at 65° C. In the interval offrom pH 5 to pH 8 the enzyme of the invention possesses a relativexylanolytic activity of more than 50%, as evidenced in FIG. 4.

The enzyme of the invention has a relative residual activity(temperature stability) after 3 hours of incubation, preferably 5 hoursof incubation, at pH 7 and 80° C. of more than 80%. After 3 hours ofincubation, preferably 5 hours of incubation, at pH 7 and 90° C., theenzyme of the invention has a relative residual activity of more than60%. After 3 hours of incubation, preferably 5 hours of incubation, atpH 7 and 100° C., the enzyme of the invention has a relative residualactivity of more than 40%.

After 24 hours of incubation at pH 7 and 80° C. a relative residualactivity of more than 60% is detectable. After 24 hours of incubation atpH 7 and 90° C. a relative residual activity of more than 40% isdetectable. After 24 hours of incubation at pH 7 and 100° C. a relativeresidual activity of more than 30% is detectable.

The enzyme of the invention has a half-life of activity (T,₁₇₈ ) ofapproximately 3 hours of boiling (100° C.), and at 90° C. the half-lifecan be estimated to approximately 24 hours.

The enzyme of the invention possesses an excellent pH stability in avery broad pH interval, namely of from below pH 5 to approximately pH12, when measured after incubation for as long as 8 hours at 65° C. Theenzyme of the invention has more than half of its relative activity inthe interval of from pH 5 to pH 10 after incubation at 65° C. for 4hours.

The enzyme of the invention is capable of hydrolysing birchwood xylansas well as oatspelt and larchwood xylans.

Immunochemical Properties

The xylanases of the invention have immunochemical properties identicalor partially identical (i.e. at least partially identical) to those of axylanase derived from the strain ATCC 43812 or the strain ATCC 43813 orthe strain DSM 4252.

The immunochemical properties can be determined immunologically bycross-reaction identity tests. The identity tests can be performed bythe well-known Ouchterlony double immunodiffusion procedure or by tandemcrossed immunoelectrophoresis according to Axelsen N. H.; Handbook ofImmunoprecipitation-in-Gel Techniques; Blackwell Scientific Publications(1983), Chapters 5 and 14. The terms "antigenic identity" and "partialantigenic identity" are described in the same book, Chapters 5, 19, and20.

Industrial Applications

Due to the excellent thermo- and pH-stability, the enzymes of thisinvention are well suited for a variety of industrial applications,including the four major applications for xylanases mentioned earlier inthis specification. The enzymes are especially well suited for treatmentof lignocellulosic pulp. Therefore, in a further aspect, the inventionrelates to the use of the xylanases for treatment of lignocellulosicpulp.

Enzymatic treatment of lignocellulosic pulp improves the bleachabilityof the pulp and/or reduces the amount of active chlorine necessary forobtaining a satisfactory bleaching.

In preferred embodiments, the xylanases of this invention can beimplemented in processes for treatment of lignocellulosic pulp asdescribed in e.g. International Patent Publications WO 91/02839 and WO92/03608.

The following examples further illustrate the present invention, andthey are not intended to be in any way limiting to the scope of theinvention as claimed.

EXAMPLE 1

Screening Example

For screening of the xylanases obtainable from the genus Rhodothermusbatch cultivation can be accomplished on the following complex medium (amodification of Medium 162 originally described by Degryse et al.[Degryse, E., Glansdorff N., Pierard A. (1978); Arch. Microbiol. 117189-196]:

    ______________________________________                                        Tryptone, Difco ®  2.5                                                    Yeast extract, Sigma ®                                                                           2.5                                                    Base solution, pH 7.2.sup.(1)                                                                        100    ml                                              Buffer solution, pH 7.2.sup.(2)                                                                      100    ml                                              ______________________________________                                    

This medium was supplemented with 2.0% NaCl and 0.5% birch xylan (Roth®7500).

    ______________________________________                                        .sup.(1) Consisting of (g/liter):                                             Nitriloacetic acid (Titriplex I)                                                                      1.0                                                   NaOH                    0.2                                                   CaSO.sub.4 ;2H.sub.2 O  0.4                                                   MgCl.sub.2 ;6H.sub.2 O  2.0                                                   Fe-citrate (0.01M)      5.0    ml                                             .sup.(2) Consisting of (g/liter):                                             KH.sub.2 PO.sub.4       5.44                                                  Na.sub.2 HPO.sub.4 ;2H.sub.2 O                                                                        21.40                                                 ______________________________________                                    

All components were sterilized by autoclaving at 121° C. for 20 min.Fe-citrate was sterilized separately.

The medium can be solidified with 2.8% (w/v) agar, Difco®.

Detection:

In order to observe extracellular xylanase activity of the colonies onagar plates, strains are streaked on solidified medium plates andincubated at 65° C. over-night. Plates are then flooded with 0.05% (w/v)aqueous solution of Congo Red for 10 minutes. After pouring off theexcess of Congo Red solution, each plate is washed twice with 1M NaClfor 10 minutes.

Xylanase producing strains are detectable from the clear zone around thethese colonies. The strains so obtained can be cultivated according tothe method described in Example 2 in order to obtain a crude xylanasepreparation.

In order to detect the activity on birchwood xylan and oatspelt xylan,respectively, 20 microlitre samples of xylanase containing culture fluidwere applied in 4 mm diameter wells in agar medium plates containing therespective xylans, the plates being incubated overnight at 55° C. andthe clearing zones visualized as described above.

Clearing zones were detected both on birch xylan and oatspelt xylan.

EXAMPLE 2

Cultivation Example

For preliminary studies and characterization of xylanases obtainablefrom Rhodothermus batch cultivation of the strain DSM 4252 wasaccomplished on a liquid complex medium of a composition as described inExample 1.

The strains were pre-inoculed for 20 hours in rotating shake-flasks on aglycerol bath at 65° C. 5 ml of this culture were used to inoculate 50ml of growth medium in 500 ml baffled shake-flasks. After 18 hours at65° C. the culture was centrifuged (on a Wifug® centrifuge at 6000 rpmfor 15 min.) and the supernatant filtrated using a 0.2μ celluloseacetate filter (Sartorius®).

These enzyme preparations were used for characterization of thexylanases of the invention.

From similar batch cultivation experiments it was demonstrated thatextracellular xylanase activity is induced by the addition of xylan.

EXAMPLE 3

Characterization Example

Assay for xylanolytic activity was performed essentially as described byBarley & Poutanen and Khan et al. [Bailey M. J, Poutanen K. (1989);Appl. Microbiol. Biotechnol. 30 5-10; and Khan A. W., Tremblay D., &Leduy Anh (1986); Enzyme Microbiol. Technol. 8 373-377].

To 1.8 ml of substrate containing 1% (w/v) birch xylan (Roth® 7500) in a25 mM sodiumphosphate buffer, pH 7.1, 0.2 ml of the enzyme preparationobtained according to Example 2 was added. After incubation for 5minutes at 65° C., 3.0 ml of 3,5-dinitrosalicylic acid (DNS) were added,and the solution was boiled for 15 minutes.

After cooling down to room-temperature the amount of colour produced wasmeasured as A₅₄₀ nm.

Temperature Dependency

In order to examine the thermal stability of the enzyme of theinvention, the enzyme preparation obtained according to Example 2 wasincubated at 80° C., 90° C., and 100° C. respectively, and samples weretaken at various intervals (1, 3, 5, and 24 hours). Enzymatic activitywas determined by the above described method, and the results arepresented in FIG. 1.

As evidenced by this Figure, the xylanase of the invention possesses aremarkable temperature stability. After 24 hours of incubation at pH 7and 80° C. a relative residual activity of more than 60% is detectable.After 24 hours of incubation at pH 7 and 90° C. a relative residualactivity of more than 40% is detectable. After 24 hours of incubation atpH 7 and 100° C. a relative residual activity of more than 30% isdetectable.

From FIG. 1 a half-life of activity (T₁₇₈ ) appears to be approximately3 hours at 100° C., and at 90° C. the half-life can be estimated toapproximately 24 hours.

In order to examine the temperature optimum of the xylanases of theinvention, the enzymatic activity was determined after incubating theenzyme preparation obtained according to Example 2 at differenttemperatures (60°-100° C.; pH 7) for 5 minutes. These results arepresented in FIG. 2.

From this Figure it appears that the enzyme of the invention possessesxylanolytic activity at temperatures of from below 60° C. to above 100°C. No pronounced temperature optimum has been detected, but apparentlythe enzyme possesses optimum activity within a broad range of from 80°to above 100° C., more specifically 85° to 100° C.

pH Dependency

In order to examine the pH stability of the enzyme of the invention, theenzyme preparation obtained according to Example 2 was incubated atdifferent pH values (5-12) at 65° C., and samples were taken at variousintervals (0, 4, and 8 hours). The xylanolytic activity was determinedby the method described above. The results are presented in FIG. 3.

From this Figure it appears that after incubation for up to 8 hours theenzyme possesses pH stability in a very broad pH interval, namely offrom below pH 5 to approximately pH 11, when measured at 65° C.

In order to examine the pH optimum of the xylanases of the invention,the enzymatic activity was determined after incubation of the enzymepreparation at different pH values (4-11) at 65° C. for 5 minutes. Theresults are presented in FIG. 4. The activity curve shown on this figureis based on the results using the following four buffer solutions:Citrate-phosphate buffer, pH 4-7; Phosphate buffer, pH 6-8; Tris buffer,pH 7-9; and Glycine-NaOH, pH 9-11.

To avoid negative interference with the DNS, the samples were dilutedwith water prior to activity determination.

From FIG. 4 it appears that the enzyme of the invention possessesxylanolytic activity in a range of from pH below 4 to above 11.Moreover, it appears that the pH optimum of the enzyme is in the rangeof from pH 5 to 7, more specifically around pH 6, as measured afterincubation for 5 minutes at 65° C. In the interval of from pH 5 to pH 8the enzyme of the invention possesses a relative xylanolytic activity ofmore than 50%.

pl of the Xylanase Activity

A powder xylanase preparation was made by lyophilizing the crude culturebroth obtained according to Ex. 2.

The pl of the xylanase activity was determined using LKB ampholine PAGplates pH 3.5-9.5 and a solution made from the lyophilized powderxylanase preparation. After the electrophoresis the gel is washed twicefor 15 minutes, once in water, once in trisbuffer pH 9, and thenoverlaid with a thin coat of detection agar consisting of 0.5% of birchxylan (Roth® 7500), 1% of agarose, pH 9. The overlaid gel is incubatedovernight at 50° C. The xylanase activity was visualized using Congo Redstaining (staining for 10 minutes with 0.1% of Congo Red and destainedfor 2×15 minutes in 1M NaCl).

At least 2 components with xylanolytic activity could be detected in therange of from 3 to 7.

We claim:
 1. A xylanase obtained from a strain of Rhodorhermus marinus, the xylanase having the following properties:(a) Optimum activity at temperatures of from 85 to 100° C.; (b) A relative activity of more than 50% in the interval of from pH 5 to pH 8 after incubation for 5 minutes at 65° C.; (c) A relative temperature stability at 80° C. of more than 80% after incubation at pH 7 for 3 hours; (d) a pH optimum of about 6; (e) a pI in the range of from 3 to
 7. 2. The xylanase according to claim 1, obtained from the strain ATCC 43812, or the strain ATCC 43813, or a mutant thereof.
 3. The xylanase according to claim 1, which hydrolyzes birchwood xylans.
 4. A process for the preparation of a xylanase having the following properties:(a) Optimum activity at temperatures of from 85° to 100° C.; (b) A relative activity of more than 50% in the interval of from pH 5 to pH 8 after incubation for 5 minutes at 65° C.; (c) A relative temperature stability at 80° C. of more than 80% after incubation at pH 7 for 3 hours; (d) a pH optimum of about 6; (e) a pI in the range of from 3 to 7;which process comprises cultivation of a xylanase-producing strain of Rhodothermus marinus in a suitable nutrient medium, containing carbon and nitrogen sources and inorganic salts, followed by recovery of the xylanase.
 5. The process according to claim 4, in which the strain ATCC 43812, or the strain ATCC 43813, or a mutant thereof, is cultivated.
 6. A process for the treatment of lignocellulosic pulp, in which the lignocellulosic pulp is treated with a xylanase obtained from Rhodochermus marinus, the xylanase having the following properties:(a) Optimum activity at temperatures of from 85° to 100° C.; (b) A relative activity of more than 50% in the interval of from pH 5 to pH 8 after incubation for 5 minutes at 65° C.; (c) A relative temperature stability at 80° C. of more than 80% after incubation at pH 7 for 3 hours; (d) a pH optimum of about 6; (e) a in the range of from 3 to
 7. 7. The process of claim 6 in which the xylanase is obtained from strain ATCC 43812, or the strain ATCC 43813, or a mutant thereof. 